Abstract:

The invention concerns the use of the fluorescence polarization phenomenon
to improve detection of fluorescent signals during a fluorescence
resonance energy transfer (FRET). In particular, the invention concerns a
method for improving signal/noise ratio in a FRET measurement. The
invention also concerns an apparatus for measuring fluorescence following
an energy transfer between a donor fluorescent compound and an acceptor
fluorescence compound in a measurement medium.

Claims:

1. Method for detecting an energy transfer between a donor fluorescent
compound and an acceptor fluorescent compound present in a measurement
medium, comprising the following steps:(i) exciting the measurement
medium by a light beam at a wavelength λ1, wherein λ1 is the
wavelength at which said donor fluorescent compound is excited,(ii)
measuring the signal resulting from the fluorescence emitted at a
wavelength λ3, wherein λ3 is the wavelength at which the
fluorescence of the acceptor fluorescent compound is emitted,(iii)
measuring the signal resulting from the fluorescence emitted at a
wavelength λ2, wherein λ2 is the wavelength at which the
fluorescence of the donor fluorescent compound is emitted, and(iv)
correcting the signal resulting from the fluorescence emitted by the
acceptor fluorescent compound at the wavelengthλ3 by the signal
resulting from the fluorescence emitted by the donor fluorescent compound
at the wavelength λ2,wherein the donor fluorescent compound and the
acceptor fluorescent compound are different,the exciting light beam is
polarized, andthe signal resulting from the fluorescence emitted at the
wavelength λ3 is measured in a plane different from the
polarization plane of the exciting light.

2. Method according to claim 1, wherein the signal resulting from the
fluorescence emitted at the wavelength λ2 is measured in a plane
different from the polarization plane of the exciting light.

3. Method according to claims 1 or 2, wherein the correction of the signal
resulting from the fluorescence emitted by the acceptor fluorescent
compound at the wavelength λ3 by the signal resulting from the
fluorescence emitted by the donor fluorescent compound at the wavelength
λ2 comprises the determination of the ratio of the signals measured
at the wavelengths λ3 and λ2.

4. Method according to claim 1, wherein the signals resulting from the
fluorescence emitted at the wavelengths λ2 and/or λ3 are
measured in a plane orthogonal to the polarization plane of the exciting
light.

5. Method according to claim 1, further comprising the following steps:(i)
exciting the measurement medium by a light beam polarized at the
wavelength λ1, wherein λ1 is the wavelength at which said
donor fluorescent compound is excited,(ii) measuring the total
fluorescence intensity (lt.sub.//).sub.λ2 emitted at the wavelength
λ2 in a plane parallel to the plane of the exciting light, wherein
λ2 is the wavelength at which the donor fluorescent compound light
is emitted,(iii) measuring the total fluorescence intensity
(lt.sub.∥).sub.λ2 emitted at the wavelength λ2 in a
plane different from the polarization plane of the exciting light,(iv)
measuring the total fluorescence intensity (lt.sub.∥)80 3
emitted at the wavelength λ3 in a plane parallel to the plane of
the exciting light, wherein λ3 is the wavelength at which the
acceptor fluorescent compound light is emitted,(v) measuring the total
fluorescence intensity (lt.sub.∥).sub.λ3 emitted at the
wavelength λ3 in a plane different from the polarization plane of
the exciting light,(vi) calculating the polarization P due to the energy
transfer between the donor fluorescent compound and acceptor fluorescent
compound according to the following formula: P = [ ( It // )
λ 3 - ( It // ) λ 2 × A ) ]
- G [ ( It ∥ ) λ 3 - ( It ∥ )
λ 2 × B ) ] [ ( It // ) λ
3 - ( It // ) λ 2 × A ) ] + nG [
( It ∥ ) λ 3 - ( It ∥ ) λ
2 × B ) ] ##EQU00002## wherein:A is the proportionality
factor between the signals resulting from the fluorescence emitted at the
wavelengths λ2 and λ3 by the donor alone, in a plane parallel
to the plane of the exciting light,B is the proportionality factor
between the signals resulting from the fluorescence emitted at the
wavelengths λ2 and λ3 by the donor alone, in a plane
different from the polarization plane of the exciting light,n=1 or 2,G is
a sensitivity correction factor specific to the measurement apparatus
used, and has a value between 0.1 and 2, and(vii) comparing the
calculated value of P with that obtained in a control measurement medium
in which the energy transfer does not take place.

11. Method according to claim 1, wherein the donor and acceptor
fluorescent compounds have a polarization greater than 50 mP.

12. Method according to claim 11, wherein the donor and acceptor
fluorescent compounds have a polarization greater than 100 mP.

13. Method according to claim 1, wherein the donor and acceptor
fluorescent compounds are selected such that following excitation at the
excitation wavelength of the donor λ1, no emission of the acceptor
is detected at the emission wavelength of the donor λ2.

14. Method according to claim 1, wherein the distance between the donor
and acceptor compounds varies as a function of biochemical events taking
place in the measurement medium.

16. Method according to claim 1, wherein the donor and acceptor compounds
are bound directly or indirectly to a hydrolyzable substrate.

17. Method according to claim 1, wherein the donor and acceptor compounds
are each bound in a covalent manner to a pair of molecules capable of
recognizing each other.

18. Method according to claim 1, wherein the donor and acceptor compounds
are each bound to two molecules capable of recognizing a third molecule
present in the measurement medium.

19. (canceled)

20. Apparatus for measuring the fluorescence resulting from an energy
transfer between a donor fluorescent compound and an acceptor fluorescent
compound present in a measurement medium comprising:means for
illuminating said medium by a polarized exciting light,means for
collecting the fluorescence emitted by the medium at different
wavelengths and in different polarization planes that are parallel or
non-parallel, to the plane of the exciting light andcomputer means for
correcting the signal measured at the emission wavelength of the acceptor
fluorescent compound by the signal measured at the emission wavelength of
the donor fluorescent compound,

21. Apparatus for measuring the fluorescence resulting from an energy
transfer between a donor fluorescent compound and an acceptor fluorescent
compound present in a measurement medium, comprising:means for
illuminating said medium by a polarized exciting light,means for
collecting the fluorescence emitted by the medium at different
wavelengths and in different polarization planes that are parallel or
non-parallel, to the plane of the exciting light andcomputer means for
calculating the polarization ofthe measurement medium specifically due to
the energy transfer taking place in the measurement medium, according to
the method described in claim 3.

22. Apparatus according to claim 20 or 21, wherein said apparatus is a
microscope.

23. The apparatus according to claim 20 or 21, wherein the different
polarization planes are orthogonal to that of the exciting light.

Description:

[0001]The invention relates to the use of the fluorescence polarization
phenomenon, in order to improve detection of fluorescence signals during
a resonance energy transfer (FRET). In particular, the invention relates
to a method for improving the signal/noise ratio in a FRET measurement.

[0002]Fluorescence resonance energy transfer (FRET) is a spectroscopic
tool widely used in the detection of biological events and in particular
of molecular interactions.

[0003]In numerous cases, the FRET, which requires bringing close together
the donor and acceptor fluorescent molecules which will be involved in
the energy transfer, proves to be a powerful tool in the detection of
biological interactions. It can be used in fields as varied as molecular
biology, the in-vitro or in-cellulo detection of enzymatic phenomena
(peptide cleavage, phosphorylation) or interactions between proteins (1,
2, 3).

[0004]Detection of the FRET phenomenon can be carried out by measuring
different parameters of the fluorescence signal emitted either by the
donor, or by the acceptor, or by both molecules. Among the most common
techniques, there can in particular be mentioned: [0005]measurement of
the reduction in the donor's fluorescence induced by the FRET phenomenon
(4), [0006]measurement of the increase in the acceptor's fluorescence
induced by the energy originating from the donor via the FRET (5),
[0007]determination of the [(acceptor fluorescence increase)/(donor
fluorescence reduction)] ratio (6), [0008]measurement of the reduction in
the lifetime of the donor's fluorescence induced by the FRET phenomenon
(7). The latter is in particular measured by the "Fluorescence Lifetime
Imaging Microscopy" (FLIM) technique, [0009]measurement of the increase
in the fluorescence of the donor involved in a FRET after the
photobleaching of the acceptor (8); this photobleaching technique is
known as Fluorescence Recovery After Photobleaching (FRAP).

[0010]Leaving aside techniques combining the FRET and time-resolved
detection made possible by the use of fluorescence donors with a long
lifetime (e.g.: HTRF), the FRET phenomenon proves complex to detect in
numerous applications based on fluorescence intensity measurements. The
need for significant energy compatibility between the donor and the
acceptor often leads to the use of molecules possessing relatively
similar fluorescence emission spectra. The resulting overlap of the
donor's and acceptor's spectra make it very difficult to precisely
measure variations in signals recorded on the donor or on the acceptor
(9).

[0011]This is particularly true when fluorescent proteins derived from
Green Fluorescent Protein (GFP) are used in the FRET experiments, such as
the Cyan Fluorescent Protein (CFP)/Yellow Fluorescent Protein (YFP)
donor/acceptor pair which is the most used. These molecules, which are
capable of being expressed in fluorescent form in numerous types of cell,
allow the detection of numerous intracellular events. However, a
significant overlap of fluorescence spectra exists between the latter,
resulting in the direct parasitic excitation of the acceptor by the donor
molecule's excitation beam. Therefore, the signal/noise ratio of the FRET
experiments carried out with this donor/acceptor pair is low, often less
than 1.5 (1). As a result, it is necessary to implement complex
experimentation protocols comprising numerous experimental controls in
order to be able to interpret the results obtained.

[0012]The technical problem to be resolved therefore involves providing a
simple and reproducible method for correcting the FRET measurement, in
particular by improving the signal/noise ratio.

[0013]It has now been found that the impact of the strong overlap of the
donor's and acceptor's fluorescence emission spectra could be
significantly reduced using the polarization properties of these
compounds in order to correct the FRET measurement.

[0014]It has been described that the appearance of an energy transfer
between two fluorescent molecules caused polarization modifications both
at the level of the donor and at the level of the acceptor: the
polarization of the donor increases when it is involved in a FRET (10),
whereas that of the acceptor involved in the FRET decreases (11).

[0015]The influence of the FRET on the relative polarization of the donor
and the acceptor has thus been used in different molecular systems in
order to detect this energy transfer between two fluorescent probes.

[0016]In particular, a homoFRET between two GFP molecules has been
detected by measuring their depolarization (12). Measurement of the
depolarization of rhodamine coupled to a lectin was used to detect a FRET
being produced between the fluorescein and the rhodamine (5). Also,
measurement of the increase in the polarization of a Concanavilin
A-Fluorescein donor made it possible to detect a FRET indicating the
formation of a molecular cluster in the lymphocyte membranes (10).

[0017]The polarization measurements used thus far therefore had the
purpose of detecting the existence of a FRET between two molecules.

[0018]Surprisingly, it has now been found that the polarized measurement
of the fluorescence signals makes it possible to better isolate the
signals emitted specifically by the donor and the acceptor involved in
the FRET and therefore to increase the signal/noise ratio in the tests
carried out.

[0019]In fact, the fluorescent proteins of GFP type for example, due to
their structure and their molecular weight, are strongly polarized
molecules. Their degree of polarization varies when they are involved in
an energy transfer: as donor molecule, their polarization increases a
little, whereas, as acceptor molecule, they undergo a strong
depolarization through the FRET phenomenon.

[0020]In a medium containing a donor fluorescent compound and an acceptor
fluorescent compound and where an energy transfer takes place between
these two compounds following the excitation of the medium at the donor's
excitation wavelength, the signal measured at the acceptor's emission
wavelength comprises: [0021]a depolarized signal originating from the
FRET (the specific signal which is to be measured), [0022]a highly
polarized signal emitted by the acceptor, as a result of its direct
excitation by the light beam intended to excite the donor (parasitic
signal), and [0023]a highly polarized signal emitted by the donor, as a
result of the excitation of the donor (parasitic signal).

[0024]The method according to the invention is therefore based on the use
of this significant variation in polarization between the donor and the
acceptor in order to improve the spectral selectivity of the FRET
measurement. In fact, according to one of the variants of the invention,
measurement of the fluorescence signal emitted is carried out either in
the polarization plane parallel to that of the polarized excitation
light, or in the polarization plane orthogonal to that of the polarized
excitation light, according to the state of polarization of the donor
molecule and that of the acceptor molecule.

[0025]The invention therefore relates to a method for detection of an
energy transfer between a donor fluorescent compound and an acceptor
fluorescent compound present in a measurement medium, in which the energy
transfer measurement selectivity is improved by using the polarization
properties of said donor and acceptor fluorescent compounds.

[0026]The energy transfer is detected by measuring the signal resulting
from the florescence emitted by the acceptor fluorescent compound at a
wavelength λ3. This emission results from the energy transfer
between a donor fluorescent compound, excited in the measurement medium
at a wavelength λ1 and said acceptor fluorescent compound.

[0027]By "measurement medium", is meant a solution comprising the donor
and acceptor fluorescent compounds; this solution can be a biological
sample, or it can contain the elements necessary for studying a
biological phenomenon.

[0028]The measurement medium can also be a sample of living tissue or
living cells, placed in an appropriate culture medium. In this case, the
donor and acceptor fluorescent compounds are present either in the
culture medium of said sample of tissue or said cells, or in the tissue
itself or in the cells.

[0029]The measurement medium can finally be constituted by a living
organism, an animal, in particular a mammal to which the donor and
acceptor fluorescent compounds have been administered. The administration
of the donor and acceptor fluorescent compounds to a living animal can be
carried out topically, by simply bringing said compounds into contact
with the animal; the donor and acceptor compounds can also be injected
into the animal; the donor and acceptor fluorescent compounds can also be
directly produced in the animal's organism by genetic engineering.

[0030]As shown in the rest of the description, the general method
according to the invention makes it possible to resolve the problems
linked to the use of donor and acceptor fluorescent compounds with low
spectral selectivity, and in particular to limit the noise linked on the
one hand to the emission of light by the donor at the emission wavelength
of the acceptor, and on the other hand to the emission of light by the
acceptor not involved in the energy transfer, the acceptor being in this
case excited directly by the exciting light.

[0031]According to a first aspect, the invention relates to a method for
detecting an energy transfer between a donor fluorescent compound and an
acceptor fluorescent compound present in a measurement medium, comprising
the following stages: [0032](i) excitation of the measurement medium by a
light beam polarized at the wavelength λ1, λ 1 being the
wavelength at which said don or fluorescent compound is excited, and
[0033](ii) measurement of the signal resulting from the fluorescence
emitted at the wavelength λ3 in a polarization plane different from
the polarization plane of the exciting light, λ3 being the
wavelength at which the fluorescence of the acceptor fluorescent compound
is emitted,said method being characterized in that moreover it comprises
the following stages: [0034](iii) measurement of the signal resulting
from the fluorescence emitted at the wavelength λ2, λ2 being
the wavelength at which the fluorescence of the donor fluorescent
compound is emitted, and [0035](iv) correction of the signal resulting
from the fluorescence emitted by the acceptor fluorescent compound at
wavelength λ3 by the signal resulting from the fluorescence emitted
by the donor fluorescent compound at wavelength λ2, in that the
exciting light is polarized, and in that the signal resulting from the
fluorescence emitted at the wavelength λ3 is measured in a plane
different from the polarization plane of the exciting light.

[0036]Measurement of the signal emitted at the emission wavelength of the
acceptor fluorescent compound in a plane different from (i.e. not
parallel to) the polarization plane of the exciting light, will make it
possible to measure the signal emitted by the strongly depolarized
species, and in particular the signal from the acceptor involved in the
energy transfer, thus reducing the part of the measured signal not
originating from the energy transfer. The plane in which the measurement
is carried out is preferentially the plane orthogonal to the polarization
plane of the exciting light. Measurements in other planes could also be
suitable.

[0037]The correction of stage (iv) above can, for example, consist of a
calculation of the ratio of the intensity of the fluorescence measured at
the wavelength λ3 to that measured at the wavelength λ2.

[0038]In the case where a measurement of fluorescence at the emission
wavelength of the donor (λ2) is carried out, measurement of the
signal resulting from the emitted fluorescence can be carried out in a
parallel or different plane, preferably orthogonal to the plane of the
exciting light.

[0039]In a second embodiment, the polarization properties of the donor and
acceptor fluorescent compounds are used in order to improve the energy
transfer measurement selectivity, in a method intended to determine the
polarization variation due to the energy transfer. As in the first method
described above, this method will make it possible to improve the
measurement selectivity, which is thus better correlated to the energy
transfer phenomenon which is to be detected.

[0040]This second embodiment comprises the following stages: [0041](i)
excitation of the measurement medium by a light beam polarized at the
wavelength λ1, λ1 being the wavelength at which said donor
fluorescent compound is excited, [0042](ii) measurement of the total
fluorescence intensity (It/.sub.//).sub.λ2 emitted at the
wavelength λ2 in the plane parallel to the plane of the exciting
light, λ2 being the wavelength at which the donor fluorescent
compound light is emitted, [0043](iii) measurement of the total
fluorescence intensity (It.sub.∥).sub.λ2 emitted at the
wavelength λ2 in a plane different from the polarization plane of
the exciting light, [0044](iv) measurement of the total fluorescence
intensity (It.sub.//).sub.λ3 emitted at the wavelength λ3 in
the plane parallel to the plane of the exciting light, λ3 being the
wavelength at which the acceptor fluorescent compound light is emitted,
[0045](v) measurement of the total fluorescence intensity
(It.sub.∥).sub.λ3 emitted at the wavelength λ3 in a
plane different from the polarization plane of the exciting light,
[0046](vi) calculation of the polarization P due to the energy transfer
between the donor fluorescent compound and acceptor fluorescent compound
according to the following formula:

in which: [0047]A represents the proportionality factor between the
signals resulting from the fluorescence emitted at wavelengths λ2
and λ3 by the donor alone, in a plane parallel to the plane of the
exciting light, [0048]B represents the proportionality factor between the
signals resulting from the fluorescence emitted at wavelengths λ2
and λ3 by the donor alone, in a plane different from the
polarization plane of the exciting light, n=1 or 2. When n=1, the term
polarization measurement is used; when n=2, it is a question of
anisotropy. [0049]G is a factor making it possible to correct the
difference in sensitivity of detection in the parallel and orthogonal
planes. This factor is either provided by the constructor, or can be
easily determined by a person skilled in the art by measuring the
polarization of substances of known polarization. In a particular
implementation, G is comprised between 0.1 and 2, preferably G is
comprised between 0.8 and 1.2, and in particular G=1; and [0050](vii)
comparison of the calculated value of P with that obtained in a control
measurement medium in which the energy transfer does not take place, a
decrease in P being indicative of an energy transfer.

[0051]According to a preferred embodiment, A and B are calculated in the
following manner:

A=(Id.sub.//).sub.λ3-(Id.sub.//).sub.λ2

B=(Id.sub.∥).sub.λ3-(Id.sub.∥).sub.λ2

(Id.sub.//).sub.λ3,(Id.sub.//).sub.λ2,
(Id.sub.∥).sub.λ3,(Id.sub.∥).sub.λ2 corresponding
to the fluorescence intensities emitted at wavelengths λ2 or
λ3, in the planes parallel to or different from the polarization
plane of the exciting light, by a measurement medium containing said
donor fluorescent compound but not containing the acceptor fluorescent
compound.

[0052]As in the first method described, the measurements carried out in a
plane different from the polarization plane of the exciting light are
preferentially carried out in the plane orthogonal to the polarization
plane of the exciting light. Measurements in other planes could also be
suitable, from the moment when the plane chosen is not the plane parallel
to the polarization plane of the exciting light.

[0053]The method according to the invention therefore makes it possible to
improve the selectivity of measurement of an energy transfer phenomenon
between a donor compound and an acceptor compound. This is particularly
advantageous in the case where the spectral selectivity between the donor
and the acceptor is not optimum, i.e. in the following cases:

[0054]case where the emission spectra of the donor and the acceptor
overlap. The methods according to the invention are particularly
effective in the case where 5 nm<λ3-λ2<100 nm,
λ3-λ2 representing the difference between the wavelengths
λ3 and λ2.

[0055]case where a direct parasitic excitation of the acceptor is possible
at the excitation wavelength of the donor (λ1).

[0056]The method according to the invention can be implemented with
numerous donor and acceptor fluorescent compounds: these compounds can be
chosen from fluorescent proteins or organic fluorophores.

[0058]The donor and the acceptor can also be organic fluorophores, for
example: rhodamines, cyanines, squaraines, fluoresceins, bodipys,
compounds of the Alexa Fluor family and their derivatives, or also the
fluorescent compounds described in the Application WO2003104685.

[0059]Finally, the donor and the acceptor can be fluorescent microspheres
or nano-crystals of the Quantom-dot type.

[0060]These fluorescent compounds and their use in the FRET systems
between a donor and an acceptor are widely described in the literature.
Moreover, a person skilled in the art is able to use the method which is
the subject of the present Application with a large number of
donor/acceptor pairs.

[0061]In a preferred aspect, the donor and acceptor fluorescent compounds
have a high polarization, in particular greater than 50 mP, preferably
greater than 100 mP. The donor and acceptor compounds the intrinsic
polarization of which is less than 50 mP can be coupled or adsorbed to
carrier molecules (organic molecules, proteins, peptides, antibodies, or
other molecules as described hereafter), which has the effect of
increasing the apparent polarization of the fluorophore and makes it
possible to use it in the methods according to the invention.

[0062]In another preferred aspect, the donor and acceptor fluorescent
compounds are chosen such that following excitation at the excitation
wavelength of the donor λ1, no emission from the acceptor is
detected at the emission wavelength of the donor λ2.

[0063]The method according to the invention therefore makes it possible to
substantially improve the detection of energy transfer phenomena, which
makes it possible in particular to study more precisely the biological
interactions.

[0064]The method according to the invention can thus be used in a
biological system in which the distance between the donor and acceptor
fluorescent compounds varies as a function of a biochemical event taking
place in the measurement medium.

[0065]In a preferred implementation, the donor and acceptor fluorescent
compounds are bound to molecules chosen from the group comprising: a
peptide, a protein, an antibody, an antigen, an intercellular messenger,
an intracellular messenger, a hapten, a lectin, biotin, avidin,
streptavidin, a toxin, a carbohydrate, an oligosaccharide, a
polysaccharide, a nucleic acid. If the donor and/or acceptor fluorescent
compounds are fluorescent proteins, they can be bound to other proteins
in the form of fusion proteins, produced by recombinant DNA techniques
well known to a person skilled in the art.

[0066]This can for example be the case if the donor and acceptor
fluorescent compounds are bound directly or indirectly to a hydrolyzable
substrate. If the measurement medium for example contains an enzyme
capable of cleaving said substrate, the detection of the evolution of the
FRET can be correlated to the enzymatic activity. It is possible to add
to such a system compounds the impact of which on the enzymatic activity
is to be studied, and observe the variation in the FRET, and therefore
the variation in the enzymatic activity, as a function of the compounds
added to the measurement medium.

[0067]By direct or indirect binding of the fluorescent compounds to the
substrate, is meant a covalent bond, optionally via spacer arms, or
non-covalent bonds by means of pairs of molecules capable of binding to
each other. Such indirect bonds include for example the case where a
fluorescent compound is bound in a covalent manner to biotin, and the
substrate comprises a streptavidin group, or also the case where the
fluorescent compound is bound to an antibody specific to a tag present on
the substrate, such as the groups 6his, flag etc.

[0068]The method according to the invention can also be implemented in
order to study the variation in a FRET in the case of an interaction
between two compounds. In this case, the donor and acceptor fluorescent
compounds are bound in a covalent manner to two molecules capable of
recognizing each other. For example, the donor compound can be bound to
an antibody or antibody fragment and the acceptor compound can be bound
to the antigen recognized by this antibody. Or also, the donor and
acceptor compounds are each bound to one member of a ligand-receptor
pair, or to two proteins interacting with each other, or also the donor
is bound to a compound regulating the activity of a protein and the
acceptor compound is bound to said protein.

[0069]Finally, the method according to the invention can also be
implemented in order to study the bond of two compounds X and Y to a
third compound Z. This can be useful for studying complex recognition
phenomena between different proteins. In this case, compound X is bound
in a covalent manner to a donor fluorescent compound, compound Y is bound
to an acceptor fluorescent compound, and an energy transfer takes place
if X and Y are bound to the molecule Z.

[0070]In a preferred aspect, the donor and acceptor fluorescent compounds
are different.

[0071]The invention finally relates to a measurement apparatus suited to
the implementation of the method according to the invention.

[0072]Such an apparatus comprises the following elements: [0073]means of
illumination of a measurement medium by a polarized exciting light, for
example, lasers, flash or continuous lamps combined with a polarizer,
[0074]means for collecting the fluorescence emitted by the measurement
medium at different wavelengths and in different polarization planes, in
particular parallel or non-parallel, preferentially orthogonal to that of
the exciting light, the detection means being able to be photomultiplier
tubes, CDD cameras or intensified cameras in front of which the
appropriate polarizers are placed, and [0075]computer means making it
possible to correct the signal measured at the emission wavelength of the
acceptor fluorescent compound by that measured at the emission wavelength
of the donor fluorescent compound, in particular computer programs
capable of calculating the ratio of the intensity of the signal collected
at the emission wavelength of the acceptor by the intensity of the signal
collected at the emission wavelength of the donor.

[0076]Another apparatus making it possible to implement the method
according to the invention comprises: [0077]means of illuminating a
measurement medium by a polarized exciting light, for example, lasers,
flash or continuous lamps combined with a polarizer, [0078]means of
collecting fluorescence emitted by the measurement medium at different
wavelengths and in different polarization planes, in particular parallel
or non-parallel, preferentially orthogonal to that of the exciting light,
the means of detection being able to be photomultiplier tubes, CDD
cameras or intensified cameras in front of which the appropriate
polarizers are placed, and [0079]computer means making it possible to
calculate the polarization of the measurement medium specifically due to
the energy transfer taking place in the measurement medium, according to
the method described above.

[0080]These apparatuses can be for example microscopes allowing
measurement of the intensity of fluorescence emitted by a sample.

[0081]The method according to the invention, its different
implementations, as well as the instruments making it possible to
implement this method make it possible to study in a precise manner the
FRET phenomena taking place in complex measurement media and in
particular biological media containing mixtures of proteins, animal or
plant cells, membranes originating from animal or plant cells or
artificial membranes.

[0082]The methods according to the invention basically making it possible
to optimize the energy transfer (FRET) measurements, they are completely
appropriate for all the techniques based on the FRET measurements.

[0083]For example, the methods according to the invention can also be
implemented in order to refine the data obtained by "FLIM" (Fluorescence
Lifetime Imaging Microscopy) type techniques. For this purpose, the
methods according to the invention are implemented using microscopy
instruments (in particular confocal microscopy systems) which make it
possible both to obtain images of the cells or biological samples
studied, and to measure the energy transfer phenomena.

[0084]Fluorescence lifetime imaging microscopy (FLIM) allows quantitative
monitoring of the FRET with great sensitivity, via the changes induced in
the lifetime of the fluorescence of the donor and/or the acceptor. More
generally, it provides access to the variations in physico-chemical
parameters in the immediate environment of the fluorescent probes.

[0085]The examples below illustrate the invention in a non-limitative
manner.

EXAMPLE 1

Determination of the Level of Polarization of Different Fluorescent
Molecules

[0092]The expression of the different fusion proteins in the HEK293 cells
was realized in the following manner:

[0093]The HEK293 cells are transitorily transfected by electroporation
with plasmids coding for different fusion proteins. The cells are then
placed at 37° C. in a regulated medium. After 24 hours, the cells
are recovered, washed in a PBS buffer, counted and fixed in a
paraformaldehyde-based solution.

[0094]All the fluorescent molecules were then distributed in black Costar
microplates in a volume of 100 μl in a PBS or PO4 (for A647)
buffer. The concentration of A647 was 10 nM. In order to measure the
level of polarization of the different fluorescent proteins, 50,000
HEK293 cells containing the different molecules were distributed in the
different wells of the microplate. The same quantity of control cells
(containing no fluorescent protein) was distributed in "control" wells.

[0095]The level of polarization was determined using a microplate
fluorescence reader, the Analyst (Molecular Devices). Depending on the
fluorescent molecule to be detected, the Analyst was equipped with the
following filters (all from Omega Optical):

[0096]The successive measurement on the same well of the fluorescences
emitted in the presence of polarizers inserted either between the
excitation source and the sample (excitation polarizer) or between the
sample and the detector (emission polarizer) will make it possible to
calculate the level of polarization of the molecules. Thus two
measurements of fluorescence intensity are carried out: [0097]the
so-called "parallel" fluorescence intensity (I.sub.//) which corresponds
to the fluorescence intensity measured with the emission polarizer
situated in the same plane as the excitation polarizer, and [0098]the
so-called "orthogonal" fluorescence intensity (I.sub.∥) which
corresponds to the fluorescence intensity measured with the emission
polarizer situated in a plane perpendicular to the excitation polarizer.

[0099]For each fluorescent molecule, the level of polarization (P) is then
obtained by means of the following formula:

P=[(I.sub.//-I.sub.∥)/(I.sub.//+I.sub.∥)]×1000

[0100]P is expressed in mP units.

[0101]Table 1 below shows the levels of polarization obtained for the
different molecules observed.

[0102]The values obtained show that the polarization of the different
fluorescent proteins used in the experiment is very high (>200 mP)
compared with that of a small organic molecule such as Alexa Fluor 647
(-17 mP).

EXAMPLE 2

Determination of the Signal/Noise (S/N) Ratio of an Intracellular FRET
Experiment

[0106]CAM fusion protein the structure of which is as follows: CFP-peptide
linker-YFP. This fusion protein was also expressed in HEK 293 cells.

[0107]The expression of these different proteins was carried out as
described in Example 1.

[0108]The cells containing CAM are those which allow the measurement of a
FRET between the CFP (donor molecule) and the YFP (acceptor molecule). In
the different so-called "positive" wells of a microplate, 25,000 cells
containing CAM, previously diluted in a PBS buffer, were distributed in a
volume of 100 μl.

[0109]In the different so-called "negative" wells of a microplate, 50,000
cells containing V1a-YFP and 50,000 cells containing CXCR4-CFP were
distributed in a PBS buffer in a total volume of 100 μl. In this case,
the absence of proximity between CFP and YFP prevents any FRET between
these two molecules and only the fluorescence noise is measured.

[0110]Two successive fluorescence measurements are carried out on the
Analyst using the following filters:

[0111]These two fluorescence measurements at 480 nm (I480 nm) or 535
nm (I535 nm) will be carried out in the presence or in the absence
of polarizers. In the presence of polarizers, only the so-called
"orthogonal" fluorescence intensity (I.sub.∥) as defined in Example
1 is measured.

[0112]Then the ratios R=(I535 nm/I480 nm) are calculated for the
positive or negative wells and that for the total fluorescence
measurements (without polarizers) or for the orthogonal fluorescence
measurement (with polarizers).

[0113]The signal/noise (S/N) of the experiment is then calculated as
follows for the measurements carried out with or without the polarizers.

(S/B)=(R535/480 positive/R535/480 negative)

[0114]The graph represented in FIG. 1 gives the signal/noise values
obtained in the absence or in the presence of polarizers.

[0115]This shows that the use of polarizers in the detection system makes
it possible to significantly increase the signal/noise ratio of the
experiment (+48%).

[0116]In fact, the so-called "orthogonal" fluorescence measurement
encourages the detection of the signal emitted by the acceptor after
energy transfer (depolarized fluorescence) compared to the fluorescence
signals emitted by fluorescence donors or acceptors not involved in a
FRET phenomenon (highly polarized fluorescence).

EXAMPLE 3

Measurement of the Degree of Polarization of the Acceptor for the
Detection of a FRET. Correction of the Contamination of the Donor in the
Measured Fluorescence Signals and Quantification of the FRET

[0117]The fusion proteins used in this example are identical to those
described in Example 2. Their expression was carried out as described in
Example 1.

[0118]50,000 cells containing CXCR4-CFP were distributed in a PBS buffer
in a total volume of 100 μl. These so-called "control" wells allow
determination of the factors A and B which will be used in order to
correct the fluorescence signal at 535 nm of the fluorescence signal
emitted by CFP at this wavelength.

[0119]In the different so-called "negative" wells of a microplate, 50,000
cells containing V1a-YFP and 50000 cells containing CXCR4-CFP were
distributed in a PBS buffer in a total volume of 100 μl. The absence
of proximity between CFP and YFP prevents any FRET between these two
molecules.

[0120]25,000 cells containing CAM allowing the measurement of a FRET
between CFP (the donor) and YFP (the acceptor), previously diluted in a
PBS buffer, were distributed in a volume of 100 μl in the different
so-called "positive" wells of a microplate.

[0121]Variable quantities of cells containing CAM and cells containing
CXCR4-CFP, previously diluted in a PBS buffer, were distributed in a
volume of 100 μl in the different so-called "contaminated" wells of a
microplate. The mixtures were produced in the following proportions:

[0122]Each series of so-called "contaminated" wells contains a variable
proportion of molecules involved in a FRET (CAM) and of free donors
(CFP).

[0123]In order to determine the degree of polarization of the acceptor,
four successive fluorescence measurements are carried out on the Analyst
using the filters and polarizers described in the table below.

[0124]For the different samples, the level of overall polarization at 535
nm (Poverall) is then obtained by means of the following formula:

Poverall=[(I535 nm//-I535 nm∥)/(I535
nm//+I535 nm∥)]×1000

[0125]P is expressed in mP.

[0126]I535 nm// is the fluorescence intensity obtained during
measurement 3 either on the positive wells or on the negative wells or on
the contaminated wells.

[0127]I535 nm∥ is the fluorescence intensity obtained during
measurement 4 either on the positive wells or on the negative wells or on
the contaminated wells.

[0128]The degree of overall polarization measured at 535 nm represents the
sum of the degree of polarization of the YFP acceptor involved in the
FRET and the degree of polarization of the CFP donor measured at 535 nm
due to the strong CFP signal contamination at this wavelength.

[0129]The determination of the degree of polarization of the YFP acceptor
involved in the FRET can be obtained by subtracting from the signals
obtained in the fluorescence measurements at 535 nm (measurements 3 and
4) the part of the signal originating from the CFP donor.

[0130]This can be carried out by establishing a proportionality between
the signal emitted by CFP at 480 nm with the different polarizers
(measurements 1 and 2) and the signal that it emits at 535 nm on the
control wells described above. For this purpose the following formulae
are used:

A=(It535 nm///It480 nm//)

B=(It535 nm∥/It480 nm∥)

[0131]It480 nm// is the mean of the fluorescence intensities obtained
during measurement 1 on the control wells.

[0132]It480 nm∥ is the mean of the fluorescence intensities
obtained during measurement 2 on the control wells.

[0133]It535 nm// is the mean of the fluorescence intensities obtained
during measurement 3 on the control wells.

[0134]It535 nm∥ is the mean of the fluorescence intensities
obtained during the measurement 4 on the control wells.

[0135]The fluorescence signals of the YFP acceptor involved in the FRET
obtained at 535 nm with the different configurations of polarizers
(If535 nm// and If535 nm 195 ) are then calculated using the
following formulae for the different samples tested:

If535 nm//=I535 nm///-(I480 nm//×A)

If535 nm∥=I535 nm∥-(I480 nm∥×B)

I480 nm/// is the fluorescence intensity obtained during measurement
1 either on the positive wells or on the negative wells or on the
contaminated wells.

[0136]I480 nm∥ is the fluorescence intensity obtained during
measurement 2 either on the positive wells or on the negative wells or on
the contaminated wells.

[0137]I535 nm// is the fluorescence intensity obtained during
measurement 3 either on the positive wells or on the negative wells or on
the contaminated wells.

[0138]I535 nm∥is the fluorescence intensity obtained during
measurement 4 either on the positive wells or on the negative wells or on
the contaminated wells.

[0139]The degree of polarization of the YFP acceptor involved in the FRET
(Pf) is then calculated for the positive, negative or contaminated wells
using the following formula:

Pf=[(If535 nm//-If535 nm∥)/(If535 nm//-If535
nm∥)]×1000

[0140]Pf is expressed in mP.

[0141]Table 2 below gives the degree of overall polarization
(Poverall) and the degree of polarization of the YFP acceptor
involved in the FRET (Pf) for the different samples:

[0142]The values shown in Table 2 above show that the formulae described
previously make it possible to recalculate from different polarized
fluorescence measurements the degree of polarization of the acceptor
involved in the FRET even if the sample contains a large quantity of CFP
donor not involved in an energy transfer.

[0143]The degree of polarization of YFP in the FRET, situated around -50
mP in our experiment, also confirms that the YFP acceptor is strongly
depolarized when it is involved in a FRET (initial polarization value 222
mP found in Example 1).